Genotoxicity is generally defined as any damage to the integrity of a cell's DNA caused either directly through chemical interaction with DNA, or indirectly through interference with the cell's structural or enzymatic machinery, that has potential to lead to an inheritable defect in the genetic information carried by that cell. The genotoxicity or DNA damage can be caused by variety of agents such as ultraviolet light, X-Rays, free radicals, methylating agents and other mutagenic compounds. These DNA damaging agents are known as genotoxins. In microorganisms such mutations may lead to the evolution of new undesirable strains of the microorganism. For instance, antibiotic or herbicide resistant bacteria may arise. In animals these mutations can lead to carcinogenesis or may damage the gametes to give rise to congenital defects in offspring.
Thus, these DNA damaging agents chemically modify the nucleotides that comprise DNA and may also break the phosphodiester bonds that link the nucleotides or disrupt association between bases (T-A or C-G). To counter the effect of these DNA damaging agents, cells have evolved number of mechanisms. For instance, the SOS response in E. coli is a well-characterized cellular response induced by DNA damage in which a series of proteins are expressed, including DNA repair enzymes, which repair the damaged DNA. The eukaryotic cells also exhibit mechanisms such as nucleotide excision repair, base excision repair, non-homologous end-joining, homologous recombination and cell cycle arrest to allow DNA repair.
Therefore, it is important to identify which agents cause or potentiate DNA damage. For instance, a method of detecting these agents may be used as a mutagenesis assay for screening compounds that are candidate medicaments, food additives or cosmetics to assess whether or not the compound of interest induces DNA damage. Alternatively, methods of detecting DNA damaging agents may be used to monitor contamination of water supplies with pollutants that contain mutagenic compounds.
Majority of the genotoxicity assays used for regulatory toxicity testing were developed in 1970's. Their throughput could not meet the requirements of the present drug discovery requirements (Krishna et al., 1998). In most of the cases, the site and mechanism by which genotoxicity is produced by the compound under the study is not known. It may happen that the target site in the test system may not be the same target site of toxic action of the new chemical entities.
Currently there are varieties of in vitro and in vivo assays to detect genotoxicity. In vitro assays include Ames test, in vitro micronucleus test, in vitro chromosomal aberration test, comet assays and mouse lymphoma assay. In vivo assays involve the measurement of the size of a tumor mass in animal models when exposed to drugs. All these above-mentioned assays require incubating the samples from several days to weeks, whereas, it is often desirable to obtain genotoxic data in a shorter time frame. The assays such as ‘Ames test’ consider the lasting DNA damage as an endpoint (either the mutated DNA or unrepaired damage in the form of fragmented DNA). However, such conditions occur only in severe cases where repair mechanism is exhausted. In most occasions, the DNA damage is repaired before such an endpoint can be measured. Hence such assays are time consuming and relatively less sensitive. Also, the involvement of animals even for the preliminary screen warrants an immediate requirement to design and implement novel tools that filter out genotoxic entities.
Some short term methods are also available for screening genotoxic compounds, such as umu test and SOS chromotest. In the umu test and SOS chromotest the host microorganism is cultured in the presence of the sample to be tested and subsequently the host microorganism is disrupted. These tests overcome the long timespan problem of the Ames test; however they have their own disadvantages. The sensitivity is low and in particular the detection sensitivity of nitroarenes and polycyclic aromatic hydrocarbons is low. In addition the detection method requires a large number of actions and additions of various reagents thereby rendering the method complicated and expensive. Due to the fact that the cell has to be disrupted in order to carry out detection of any induction it is only possible to carry out one measurement on the cell.
Exposure of cells to genotoxic agents results in the regulation of number of damage response genes. The change in the expression of such genes upon DNA damage can hence be used to develop assays that detect early genotoxic responses in the cell. Among the well characterized events induced by genotoxic stress is the activation of p53 signaling pathway. The tumor suppressor gene p53 maintains genomic stability by inducing cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1gene promoter as an early response to DNA damage. It has been suggested that p53 activates expression of its target genes such as p21WAF1/Cip1 and GADD by multiple but perhaps interrelated mechanisms.
Treatment of mammalian cells with genotoxic agents causes an increase in the mRNA levels of a number of ‘damage response’ genes (reviewed by Holbrook and Fornace). Many of these genes are also inducible by phorbol ester treatment. Among those that do not respond to phorbol ester treatment, some can be activated by the tumor suppressor gene p53, such as WAF1 and GADD45. However, there are also a number of DNA damage inducible genes for which the activation signal is unknown, and GADD153 is one of these genes. GADD153 is of particular interest because the magnitude of the increase in GADD153 mRNA following cellular injury is greater than most other ‘damage response’ genes.
GADD153 was originally cloned by subtractive hybridization of W-treated versus proliferating Chinese hamster ovary cells. The GADD153 gene was one of a subset of genes that was induced by UV-radiation and other forms of DNA damage, but not by heatshock or phorbol ester treatment. This subset of genes was found to be coordinately regulated by a number of agents that damage DNA or induce cell cycle arrest. GADD153 is highly conserved among mammalian species; hamster GADD153 shares 78% nucleotide sequence identity with the human exons (Park et al., 1992) and >85% with the mouse exons (Ron and Habener, 1992). Gadd153 is induced by DNA damaging agents in cells with mutant or absent p53 and the induction is greater in p53 wt cells (Hollander and Fornace, 1995). It has been shown previously that hamster GADD153 gene promoter over expressed in mammalian cells showed better relative fold induction when treated with MMS and TPA (Luethy et al., 1990).
U.S. Pat. No. 6,344,324 discloses a recombinant DNA molecule comprising the regulatory element of the hamster GADD153 upstream promoter region that activates gene expression in response to a wide range of cellular stress conditions, linked to a DNA sequence that encodes GFP (Green Fluorescent Protein). This reporter system is carried out in a human head and neck squamous-cell carcinoma cell line. However, problems associated with this reporter system are that it requires at least a four day treatment period at test agent concentrations that result in less than 10% cell survival, followed by analysis of fluorescence by flow cytometry. In addition, the biological relevance of any gene induction when tested with agents at this level of toxicity is debatable. Furthermore, this development does not disclose a means of specifically monitoring the presence of agents that may cause or potentiate DNA damage, and the mechanism of GADD153 induction remains unclear. Hence, this system is of very limited use as a human DNA damage biosensor.
Another patent, WO2010/112821 discloses a DNA sequence encoding Gaussia luciferase (GLuc) reporter protein which is operatively linked to a human GADD45a gene promoter and a human GADD45α gene regulatory element. This is designed to activate expression of the DNA sequence encoding Gaussia luciferase (GLuc) reporter protein in response to genome damage. The advantage of this system is that it does not require the lysis of cells during the assay as GLuc is secretory. An advantage of using luminescence as a reporter assay is that there is no need for the incident light, as required in fluorescence based assays. Hence, unwanted fluorescence can be avoided that would otherwise mask the signal from the GFP reporter protein. The use of luminescence therefore permits screening colored and fluorescent test compounds. The above mentioned assays are limited to being a screen capable of identifying test compounds that trigger a particular DNA damage response pathway.
For the foregoing reasons, there is a need for high throughput in vitro assay which can detect wide range of genotoxins in eukaryotic mammalian cells, by retaining the simplicity associated with chemiluminescent assay. The present invention employs three genotoxicity early response gene promoters in a single cell which makes this invention more sensitive and efficient compared to other currently available assays.